The long-term goal of this research project is to study the role of AIF in cardiac apoptosis. Although the caspases are thought to be central mediators of the apoptotic program, our preliminary studies have demonstrated that adult cardiomyocytes are both resistant to caspase-dependent apoptosis, and exhibit a significant activation of apoptosis inducing factor (AIF), a prototypical factor involved in caspase-independent apoptosis, under apoptotic stimulation. The role of caspase-independent apoptosis in heart is poorly understood. However, we have found that transgenic mice with cardiac-specific expression of the caspase inhibitor, CrmA, exhibit significant AIF activation after ischemia/reperfusion (I/R). As a result, despite the complete inhibition of caspase activity, there is only a partial inhibition of myocardial damage. However, the concurrent inhibition of PARP-1 inhibits AIF activation and provides more effective cardioprotection after I/R in CrmA mice. These findings suggest that AIF-induced apoptosis may have a significant role in causing cell death in cardiovascular insults. Our hypothesis is that AIF is a principal mediator of caspase-independent pathways in cardiac apoptosis, and that the inhibition of AIF activation will be important for achieving a complete and effective anti-apoptotic therapy in heart. In Specific Aim 1, we will examine the presence of and conditions for AIF-induced cardiac apoptosis in vitro. First, we will investigate whether apoptotic stimuli known to cause caspase-dependent apoptosis also activate AIF. Next, we will determine if the activation of AIF is increased in the setting of caspase inhibition. In Specific Aim 2, we will investigate the regulation of AIF-induced cardiac apoptosis and inhibitory strategies in cardiomyocytes in vitro. First we will examine how the subcellular localization of AIF is related to its apoptotic properties. In these experiments, we will generate AIF deficient adult cardiomyocytes by overexpressing siRNA sequence specific for rat AIIF, and use them to examine the effect of reconstituting AIF mutants that possess specific subcellular characteristics. Then, we will determine whether PARP-1 activation is necessary for AIF release in cardiomyocytes. In Specific Aim 3, we will identify the role of AIF-induced apoptosis in heart, and identify effective strategies for inhibiting AIF-induced apoptosis in vivo. We will first examine the contribution of AIF-induced apoptosis during I/R in mouse heart in vivo. We will then explore a mechanism and a strategy for inhibiting AIF activation using PARP-1 inhibition in I/R-induced cardiac apoptosis in vivo.